PROJECT SUMMARY This proposal describes a five-year career development program to prepare Dr. Churko for a career as an independent investigator. This program builds on Dr. Churko's background as a cell and molecular biologist by providing him with expertise in gene editing and sequencing technology to advance our understanding of notch signaling within the heart. Dr. Churko's mentor is Dr. Joseph Wu, a Professor of Medicine/Cardiology and Director of the Stanford Cardiovascular Institute at Stanford University. Dr. Wu is an excellent mentor with extensive experience in hiPSC derivation, differentiation into cardiomyocytes (hiPSC-CMs), and using hiPSC-CMs to study cardiovascular development and disease. The K99 phase will consist of structured mentorship by the primary mentor, complementary meetings with the advisory committee, formal coursework, a provocative research project, and a program of career transition. The K99 phase of Dr. Churko's studies will focus on supporting Dr. Churko's RNA-seq data and further develop tools (CRISPR-Cas9 notch knock- out lines) to comprehensively study notch signaling at a whole genome level. In Dr. Churko's preliminary data, he has observed a striking enrichment in notch-specific genes and transcription factors only found within the adult human ventricles. This finding is also supported in mouse and chick studies which demonstrated that knocking out members of the notch pathway cause atrial genes to be expressed within the ventricles. With the current hiPSC technology to study human cardiomyocytes in culture, combined with advancements in high-throughput sequencing technology, Dr. Churko is in a unique position to uncover unknown regulatory and signaling events which define the gene signatures of the human ventricles. In AIM1 of Dr. Churko's proposal, he will develop a PCR panel of genes which are specifically expressed in the different chambers. This panel will be a valuable contribution to the cardiac community because it will allow researchers to define and assess pathways which regulate the development and identify of each heart chamber. Dr. Churko will use this panel to assess and verify which small molecules and ligands promote the ventricular gene signature, and, in using CRISPR-Cas9 technology outlined in AIM2, which notch family members are necessary for this signature. Dr. Churko will then verify these markers as being cardiac specific by immunofluorescence and quantify these differences within the heart by mass spectroscopy. Further, in AIM1, Dr. Churko will be the first to study notch-specific signaling in cardiomyocytes at a single-cell level (using single-cell Western blotting). Finally, in AIM3, Dr. Churko will assess the function and transcription changes which occur from notch signaling in cardiomyocytes. He will perform RNA-seq on hiPSC-CMs with activating notch signaling and identify DNA binding patterns and motifs specific to notch signaling (NOTCH1-ICD, HEY2). A unique goal in this aim is to integrate both RNA-seq data with ChIP-seq data to experimentally verify which transcription factor binding events lead to changes within cardiomyocyte gene signatures. Collectively, the proposed work will provide a comprehensive picture of notch signaling within human cardiomyocytes. In addition, this work will provide the foundation for future studies on engineering hiPSC-CMs into expressing ventricular and atrial gene signatures, which will be carried out by Dr. Churko as an independent investigator.